Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases |
| |
| Institution: | 1. Dana-Farber Cancer Institute, Boston, MA, USA;2. Vanderbilt-Ingram Cancer Center, Nashville, TN, USA;3. Peninsula Oncology Centre, Melbourne, VIC, Australia;4. Memorial Sloan Kettering Cancer Center, New York, NY, USA;5. The Royal Marsden Hospital, London, UK;6. Hospital Arnau de Vilanova, Lleida, Spain;7. Sarah Cannon Research Institute, and Tennessee Oncology, Nashville, TN, USA;8. Palacky University Medical School and Teaching Hospital, Olomouc, Czech Republic;9. University of Alabama, Birmingham, AL, USA;10. National University Hospital, Singapore;11. Royal Melbourne Hospital, Parkville, VIC, Australia;12. Masaryk Memorial Cancer Institute, Brno, Czech Republic;13. Herlev University Hospital, Copenhagen, Denmark;14. Mayo Clinic, Jacksonville, FL, USA;15. Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium;p. Washington University School of Medicine, St Louis, MO, USA;q. Genentech, South San Francisco, CA, USA;r. Barts Cancer Institute, Queen Mary University of London, London, UK;1. Department of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey;2. Department of Medicinal Laboratory, Vocational School of Health Services, Selcuk University, Konya, Turkey;3. Department of Analytical Chemistry, Pavol Jozef ?afárik University in Ko?ice, Ko?ice, Slovakia;4. Department of Chemistry, University of Nyíregyháza, Nyíregyháza, Hungary;5. Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, Nyíregyháza, Hungary;6. Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit, Mauritius;1. Chemical Engineering Department, Universiti Teknologi PETRONAS, 32610, Bandar Seri Iskandar, Perak Darul Ridzuan, Malaysia;3. Institute of Petroleum Engineering, School of Energy, Geoscience, Infrastructure and Society, Heriot-Watt University Malaysia, No 1 Jalan Venna P5/2, Precinct 5, 62200, Putrajaya, Malaysia;1. Chemical Engineering Department, Universiti Teknologi PETRONAS, 32610 Bandar Seri Iskandar, Perak Darul Ridzuan, Malaysia;1. Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece;2. Division of Immunology, Biomedical Sciences Research Center ‘Alexander Fleming’, Athens 16672, Greece |
| |
| Abstract: | The enantioselective microbial reduction of 6-oxo-8-4-4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-4-4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP+-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD+-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
